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HEMATOPOIESIS
Cell cycle exit during terminal erythroid differentiation is associated
with accumulation of p27Kip1 and inactivation of cdk2 kinase
Fen F. Hsieh, Lou Ann Barnett, Wayne F. Green, Karen Freedman, Igor Matushansky, Arthur I. Skoultchi, and Linda L. Kelley
Progression through the mammalian cell
cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclindependent kinase inhibitors (CKIs). The
function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins
p21Cip1 and p27Kip1 during erythropoietininduced terminal differentiation of primary erythroblasts isolated from the
spleens of mice infected with the anemiainducing strain of Friend virus was investigated. Both p21Cip1 and p27Kip1 proteins
were induced during erythroid differentia-
tion, but only p27Kip1 associated with the
principal G1 CDKs—cdk4, cdk6, and cdk2.
The kinetics of binding of p27Kip1 to CDK
complexes was distinct in that p27Kip1
associated primarily with cdk4 (and, to a
lesser extent, cdk6) early in differentiation, followed by subsequent association
with cdk2. Binding of p27Kip1 to cdk4 had
no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2
kinase activity was associated with p27Kip1
binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G1
growth arrest. Inhibition of cdk4 kinase
activity late in differentiation resulted from
events other than p27Kip1 binding or loss
of cyclin D from the complex. The data
demonstrate that p27Kip1 differentially
regulates the activity of cdk4 and cdk2
during terminal erythroid differentiation
and suggests a switching mechanism
whereby cdk4 functions to sequester p27Kip1
until a specified time in differentiation when
cdk2 kinase activity is targeted by p27Kip1
to elicit G1 growth arrest. Further, the data
imply that p21Cip1 may have a function independent of growth arrest during erythroid
differentiation. (Blood. 2000;96:2746-2754)
© 2000 by The American Society of Hematology
Introduction
Cell differentiation imparts unique identity through a coordinated
tissue-specific gene expression program. This program is tightly
coordinated with cell cycle exit. Commitment to cell division is
regulated by a family of G1 cyclin-dependent protein kinases
(CDKs) (reviewed in Morgan1 and Pavletich2). CDKs are regulated
by activating proteins, cyclins, (reviewed in Roberts3) and cyclindependent kinase inhibitors (CKIs), which either block or enhance
the activity of the cyclin-CDK complexes (reviewed in Sherr and
Roberts4 and Nakayama and Nambe5). The precise role these
proteins play in establishment and maintenance of the nonproliferative
state associated with terminal differentiation has not been determined.
Two families of CKIs regulate the function of CDKs and are so
designated based on their structure and CDK targets (reviewed in
Sherr and Roberts4). The INK4 family consists of 4 proteins
(p16INK4a, p15INK4b, p18INK4c, and p19INK4d) that specifically inhibit
the catalytic subunits of cyclin D-dependent kinases (cdk4 and
cdk6)6. The Cip/Kip family consists of 3 members (p21Cip1, p27Kip1,
and p57Kip2). Mixing experiments in vitro and overexpression
studies suggested that Cip/Kip proteins ubiquitously inhibit the
activities of cyclin D-, E-, and A-dependent kinases, but more
physiological models challenge this conclusion. Cip/Kip specificity
for cyclin D and E complexes during the irreversible growth arrest
associated with terminal differentiation has not been described.
Murine erythroid progenitor cells infected with the anemia-
inducing strain of Friend virus represent a model system in which
to study the molecular events associated with terminal erythroid
differentiation in response to erythropoietin (EPO). Infection of
mice with a retroviral complex consisting of replication-defective
spleen focus-forming virus and replication-competent Friend murine leukemia virus leads to polyclonal expansion of proerythroblasts in the spleens of susceptible mice due to expression of the
unique spleen focus-forming virus protein gp55 (reviewed in
Ben-David and Bernstein7). Gp55 binds to and partially activates
the EPO receptor,8,9 resulting in the expansion of nontransformed
proerythroblasts (FVA erythroblasts), dependent on EPO for growth
in vivo and in vitro. FVA erythroblasts cultured in vitro with EPO
for 48 hours undergo a transient proliferative burst followed by
withdrawal from the cell cycle and accumulation into the G1
phase.10,11 Coincident with cell cycle arrest, FVA erythroblasts
undergo a program of terminal differentiation characterized by
expression of ␤-globin mRNA, hemoglobin production, nuclear
condensation, and enucleation.12-14 The molecular mechanisms
associated with FVA erythroblast terminal differentiation and cell
cycle exit are unknown.
We examined the regulation and function of members of the
Cip/Kip and INK4 families of CKIs to determine their role in
growth arrest of terminally differentiated erythroid cells. In the
Cip/Kip family we limited our study to p21Cip1 (p21) and p27Kip1
From the Departments of Pathology/Division of Cell Biology and Immunology
and Medicine/Division of Hematology, University of Utah School of Medicine
and Huntsman Cancer Institute, Salt Lake City, UT; and the Department of Cell
Biology, Albert Einstein College of Medicine, Bronx, NY.
grant NCI 5P30CA42014.
Reprints: Linda L. Kelley, Department of Medicine, University of Utah School
of Medicine AR159, 50 North Medical Dr, Salt Lake City, UT 84132; e-mail:
[email protected].
Submitted March 17, 2000; accepted June 16, 2000.
Supported by the American Cancer Society, The Primary Children’s Medical
Center Research Foundation, National Institutes of Health grants P50DK49219
(L.L.K., from the Center of Excellence in Molecular Hematology), T32DK07115
(L.A.B.), and CA163687 (A.I.S), and Utah Regional Cancer Center core
2746
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2000 by The American Society of Hematology
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
(p27). Expression of p57Kip2 was not evaluated because p57Kip2
protein is restricted to a limited number of tissues, primarily during
development,15,16 and was not detected in mouse spleens.15 Here
we report that p19INK4d (p19), p21, and p27 proteins are induced
during terminal erythroid differentiation and exhibit differential
specificities for G1 CDKs, leading to distinct functions. The data
provide a model for the roles of p27, cdk4, and cdk2 in cell cycle
exit during terminal differentiation.
Materials and methods
Cell isolation and culture
Nucleated erythroblasts were isolated from spleens of 8- to 12-week-old
Balb-c mice infected 2 weeks previously with 104 spleen focus-forming
units of the anemia-inducing strain of Friend virus (FVA), as described.17,18
Briefly, splenocytes were separated by velocity sedimentation at unit
gravity on a continuous 1% to 2% bovine serum albumin (BSA) gradient.
Purified erythroblasts were cultured in Iscove modified Dulbecco medium
(IMDM) containing 1% BSA, 30% fetal bovine serum (FBS) (HyClone
Laboratories, Logan, UT), 100 U/100 g per milliliter penicillin/streptomycin (Gibco BRL, Gaithersburg, MD), and, where indicated, 1 U/mL pure
recombinant human erythropoietin (EPO) (Ortho Pharmaceutical, Raritan,
NJ). EPO-independent murine erythroleukemia cells (MEL)19 were maintained in IMDM supplemented with 10% FBS and 100 U/100 g per
milliliter penicillin/streptomycin. The EPO-dependent HCD57 cell line was
a gift of Dr David Hankins and was maintained in IMDM containing 30%
FBS, 100 U/100 g per milliliter penicillin/streptomycin, and 1 U/mL EPO.
A retinoblastoma (Rb)⫹ T-lymphocyte cell line, Molt 4,20 was maintained
in RPMI supplemented with 10% FBS. An Rb⫺ human breast carcinoma
cell line, MDA MB436,21 was the gift of Dr Maria Frexes (Vanderbilt
University, Nashville, TN) and was maintained in McCoy medium supplemented with 10% FBS. NIH-3T3 cells (ATCC, Rockville, MD) were
maintained in Dulbecco modified Eagle medium with 4.5 g/L glucose
supplemented with 10% FBS.
Morphologic analyses
Cytocentrifuge (Shandon, Pittsburgh, PA) slide preparations of cells were
stained with 3,3⬘-dimethoxybenzidine, counterstained with hematoxylin
and photographed using oil immersion optics.
p27Kip1 INHIBITION OF cdk2 KINASE IN ERYTHROID
2747
tions were performed at 42°C for 16 hours using 106 cpm of the labeled
probe per milliliter hybridization buffer. Blots were washed twice with
2 ⫻ SSC, 0.5% SDS at room temperature for 10 minutes each and twice
with 0.1 ⫻ SSC, 0.1% SDS at 55°C for 40 minutes each. Full-length
Mo-MLV DNA23 was used to probe for the detection of murine retroviral
sequences. The mouse ␤-globin probe was a PstI fragment containing the
first 2 exons of the ␤-major globin gene.24 The cdk2 probe was a full-length
human EcoRI fragment (Dr James Whitlock, Vanderbilt University). The
cdk4 probe was a full-length human PstI/ BamHI fragment (Dr Steven
Hanks, Vanderbilt University). The p21 probe was a XhoI fragment
containing the entire murine cDNA (Dr David Beach, Cold Spring Harbor,
NY). The cyclin D3 probe was an EcoRI fragment corresponding to the
carboxyl terminal portion of the coding sequences. The p18 and p19 probes
were BamHI/EcoRI fragments, and the p15 and p16 probes were EcoRI
fragments containing the entire coding sequences. Dr Charles Sherr (St.
Jude’s Research Hospital, Memphis, TN) provided the cyclin D3, p18, p19,
p15, and p16 cDNA.
Immunoblot analysis of CDK and CKI proteins
Cells were cultured under the indicated conditions and washed in PBS, and
cell pellets were prepared. Pellets were snap frozen in an ethanol–dry ice
slurry and stored at ⫺70°C until further analysis. Cell lysates were prepared
by resuspending cell pellets in boiling 2⫻ sample buffer containing 83
mmol/L Tris-HCl (pH 6.8), 20% glycerol, 10% SDS, 1.28 mol/L mercaptoethanol, and 0.01% bromophenol blue. Lysis was facilitated by sonication.
Approximately 150 ␮g of each whole cell lysate was electrophoretically
separated on a 12% polyacrylamide gel. Proteins were transferred to
polyvinylidene difluoride (PVDF) membranes (Bio-Rad) and blocked
overnight at 4°C in blocking buffer containing 20 mmol/L Tris (pH 7.5),
137 mmol/L NaCl, and 0.1% Tween 20 (TTBS) containing 5% nonfat dry
milk (NFDM). Immunoblotting was performed using either 1 ␮g (p21, p27,
p18, p19) or 0.1 ␮g (cdk2, cdk4, cdk6, cyclin D3) antibody per milliliter
TTBS with 5% NFDM for 2 hours at room temperature. All antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary
antibodies included a 1:10 000 dilution in TTBS containing 5% NFDM of
antimouse IgG (NA931; Amersham Life Science, Buckinghamshire, UK)
(p21) or antirabbit IgG (A-6154; Sigma) (p27, cdk2, cdk4, cdk6, cyclin D3,
p18, p19) conjugated to horseradish peroxidase (HRP) and allowed to
incubate for 1 hour at room temperature. Bound antibodies were detected by
enhanced chemiluminescence (Amersham).
Immunoblot analysis of Rb proteins
Flow cytometric DNA analysis
Single parameter analysis of DNA content was performed on cells freshly
removed from culture, fixed in methanol, and resuspended in a staining
solution of 500 ␮L RNase (200 U/mL) (Sigma, St Louis, MO) and 500 ␮L
propidium iodide (PI) (50 g/mL) (Molecular Probes, Eugene, OR). Linear
fluorescence signals of PI (area and width) were assessed on a Becton
Dickinson FACScan flow cytometer with dye excitation by 15 mW 488 nm
laser light. Data were stored as list mode files of at least 50 000 single cell
events for subsequent off-line analysis using Modfit and WinList software
(Verity Software, Topsham, ME). DNA cell cycle analysis was accomplished using the DIP_N2 and DIP_N3 algorithms in Modfit.
Northern blot analysis
Northern blot analysis was performed as previously described.10 Briefly,
total RNA was isolated from cells by a single-step guanidinium thiocyanate–
phenol method using Ultraspec RNA (Biotex Laboratories, Houston, TX)
and electrophoretically separated in 1% agarose gels containing 0.66 mol/L
formaldehyde. After electrophoresis, the ethidium bromide-stained gels
were photographed and blotted onto nylon membranes (Bio-Rad Laboratories, Hercules, CA). Prehybridization and hybridization were performed in
50% formamide, 6 ⫻ SSC (1.5 mol/L NaCl, 150 mmol/L sodium citrate),
1 ⫻ Denhardt solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
BSA), and 0.5% sodium dodecyl sulfate (SDS). All probes were radiolabeled by random priming22 using [-32P]dCTP (800 Ci/mmol). Hybridiza-
Freshly isolated cells were washed in PBS and resuspended in lysis buffer A
containing 50 mmol/L HEPES (pH 7.0), 250 mmol/L NaCl, 0.1% NP40, 25
mol/L NaF, 200 mol/L Na3VO4, 1 mmol/L phenylmethylsulfonyl fluoride
(PMSF), and 5 ␮g/mL aprotinin for 30 minutes on ice. Lysis was facilitated
by vortexing at 10-minute intervals. Lysates were cleared by centrifugation,
and 50 ␮g protein was electrophoretically separated on 7% polyacrylamide
gels and transferred to nitrocellulose membranes. Membranes were blocked
for 1 hour at room temperature in TTBS containing 5% NFDM. Immunoblotting was performed with 1 ␮g anti-Rb (PMG3-245; PharMingen, San
Diego, CA) per milliliter TTBS blocking buffer at 37°C for 2 hours,
followed by a 1:2000 dilution of antimouse IgG conjugated to HRP
(A-4416; Sigma) for 1 hour at room temperature. Where indicated, gp55
protein was detected with a 1:500 dilution of rat monoclonal antibody 7C10
(Dr Sandra Ruscetti, National Cancer Institute, Frederick, MD) per
milliliter blocking buffer for 1 hour at 37°C, followed by a 1:5000 dilution
of goat antirat HRP (A-9037; Sigma). Bound antibody was detected using
enhanced chemiluminescence.
Immunoprecipitation of CDK and CKI proteins
Cells were removed from culture at the indicated times, washed in PBS, and
centrifuged at 1500 rpm for 5 minutes at 4°C. Cell pellets were immediately
snap frozen in an ethanol–dry ice slurry and stored at ⫺70°C until further
analysis. Frozen cell pellets were resuspended in 1.2 mL lysis buffer B
containing 50 mmol/L Tris (pH 7.4), 300 mmol/L NaCl, 2 mmol/L EDTA,
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2748
HSIEH et al
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
50 mmol/L sodium fluoride, 0.5% NP-40, 1 mmol/L sodium vanadate,
1 mmol/L PMSF, and 10 ␮g/mL each of leupeptin, aprotinin, trypsin
inhibitor, and pepstatin A. Whole cell lysates were incubated for 30 minutes
on ice, and lysis was facilitated by three 10-second cycles of sonication.
Lysates were centrifuged at 10 000 rpm for 5 minutes at 4°C. Cell
supernatants were removed and stored on ice. Remaining nuclear pellets
were resuspended in 200 ␮L lysis buffer B and were incubated on ice for 15
minutes. Nuclear lysates were centrifuged as above, and the supernatant
was combined with cellular supernatant. Pooled supernatants were centrifuged at 14 000 rpm for 20 minutes at 4°C. Lysates containing 350 ␮g
protein were incubated with 2 ␮g of the indicated antibodies at 4°C for 2
hours. Protein A agarose (30 ␮L) was added to each sample and allowed to
rotate for 2 hours at 4°C. Mixtures were centrifuged at 11 000 rpm for 5
minutes at 4°C. Agarose pellets were washed 4 times with buffer B and
resuspended in 25 ␮L boiling 2⫻ sample buffer, snap frozen, and stored at
⫺70°C until further analysis. Frozen pellets were boiled for 5 minutes and
electrophoretically separated on 12% polyacrylamide gels. Proteins were
transferred and analyzed by immunoblot analysis as described above.
Rb kinase assay
Rb kinase assays were performed as described by Matsushime et al.25
Briefly, cell pellets were resuspended in lysis buffer C containing 50
mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 2.5 mmol/L EGTA, 1 mmol/L
EDTA, and 0.1% Tween 20 containing 10% glycerol, 1 mmol/L dithiothreitol (DTT), 0.1 mmol/L PMSF, 0.2 U/mL aprotinin, 10 mmol/L glycerophosphate, 0.1 mmol/L sodium vanadate, 1 mmol/L sodium fluoride, and 10 ␮g
leupeptin. Whole cell lysates were sonicated at 4°C 3 times for 10 seconds
each, followed by centrifugation at 10 000g for 5 minutes. Immunoprecipitations were performed by incubating lysates containing 1 mg protein with
4 ␮g antibodies (cdk2, cdk4, cdk6) for 4 hours at 4°C followed by a 2-hour
incubation with protein A-Sepharose. Immunoprecipitates were washed 4
times with 1 mL buffer C and 2 additional times with kinase buffer D
containing 50 mmol/L HEPES (pH 7.5), 10 mmol/L MgCl2, and 1 mmol/L
DTT. The beads were suspended in 30 ␮L reaction buffer D containing 2.5
mmol/L EGTA, 1 mmol/L DTT, 20 mol/L adenosine triphosphate (ATP), 10
mmol/L glycerophosphate, 0.1 mmol/L sodium vanadate, 1 mmol/L sodium
fluoride, 2 ␮g Rb substrate freshly prepared from an Rb GST fusion
protein,25 and 10 ␮Ci (␥-32P) ATP (6000 Ci/mmol; NEN Life Science,
Boston, MA) at 30°C for 30 minutes. The reaction was terminated by
adding 2.5⫻ SDS sample buffer. Samples were boiled for 5 minutes and
electrophoretically separated on 12% polyacrylamide gels. Dried gels were
exposed to x-ray film and examined by autoradiography.
Results
FVA erythroblasts undergo G1 growth arrest during
EPO-mediated terminal differentiation
Freshly isolated FVA erythroblasts were cultured in the presence of
EPO and analyzed for DNA content and differentiation status
(Figure 1). Approximately 32% of freshly isolated erythroblasts
were in G1 phase at the initiation of culture. After culture in the
presence of EPO, the percentage of cells in G1 phase increased to
49% and 84%, respectively, at 24 and 48 hours. Benzidine stains
were prepared on cytospin slides at the same time points and demonstrated that cells were developmentally immature at the initiation of
culture, as determined by the lack of hemoglobin production. By 24
hours cells had initiated hemoglobin production and nuclear
condensation, and by 48 hours most cells had enucleated.
Multiple cell cycle–associated mRNAs are transcriptionally
regulated during erythroid differentiation
Initially, experiments were performed to determine which of the
principle G1 CDK, cyclin, and CKI regulators were present in
Figure 1. DNA cell cycle kinetics and morphologic changes during terminal
erythroid differentiation. FVA erythroblasts were cultured for 0 (A, D), 24 (B, E), or
48 (C, F) hours with EPO. Cells were removed from culture and analyzed for DNA cell
cycle analysis of PI-stained cells by flow cytometry (A, B, C) or benzidine staining (D,
E, F). Percentages of cells in G1 at 0, 24, and 48 hours of culture were 32%, 49%, and
84%, respectively.
differentiating erythroblasts and to correlate their expression with
an erythroid-specific differentiation marker, ␤-globin. Northern
blot analyses of total RNA from cells cultured in the presence of
EPO are shown in Figure 2. Ethidium bromide–stained ribosomal
RNA and expression of viral mRNAs are shown as loading and
blotting controls, respectively. Globin mRNA expression increased
during the time course and served as evidence that the cells were
differentiating. Levels of cdk2, cdk4, and cyclin D3 mRNA
decreased during differentiation, albeit with different kinetics.
Cyclin D1 and D2 mRNA were not expressed in differentiating
erythroblasts at any time point (data not shown). As previously
shown,11 the expression of p21 mRNA increased significantly from
0 to 42 hours of culture. These data suggested that cell cycle arrest
might occur from the down-regulation of positive regulators of G1
as well as the up-regulation of G1 inhibitors. However, because the
regulation of many cell cycle–associated gene products is known to
occur at the posttranscriptional level, we shifted our focus to the
study of protein expression.
Cip/Kip proteins are induced during erythroid differentiation
Steady state levels of proteins isolated from erythroblasts cultured
with EPO are shown in Figure 3. Erythroblasts lacking p21 were
included as a negative control for p21, and p21 wild-type erythroblasts, treated with the DNA damaging agent actinomycin D
(ActD)—which activates wild-type p53 leading to enhanced production of p21 protein10,11—served as a positive control for p21. Levels
of p21 protein increased during differentiation, peaking at 24 hours
and declining by 48 hours. No p21 protein was detected in p21 null
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
p27Kip1 INHIBITION OF cdk2 KINASE IN ERYTHROID
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erythroid differentiation suggested they might function in growth
arrest by binding to and inhibiting one or more G1 CDKs.
Endogenous Rb accumulates in a hypophosphorylated form
during erythroid differentiation
Inhibition of G1 CDK function in vivo should prevent phosphorylation of their common substrate, the retinoblastoma (Rb) protein
(reviewed in Mulligan and Jacks27). We examined the phosphorylation status of endogenous Rb by observing the migration pattern of
Rb proteins using standard gel electrophoresis and Western blot
analysis (Figure 4). Rb migrated as 2 species in undifferentiated
erythroblasts, a predominant hyperphosphorylated (ppRb) species
of approximately 117 kd and a less abundant hypophosphorylated
(pRb) form of approximately 110 kd. After 24 hours in culture with
EPO, pRb and ppRb were present in approximately equal amounts.
After 48 hours of culture, when cells were maximally growth
arrested into G1 (Figure 1C), all the Rb protein had accumulated in
the hypo-phosphorylated (pRb) form. Hypophosphorylated Rb did
not accumulate in cells cultured in the absence of EPO, indicating
that differentiation was required for pRb accumulation. Known
Rb⫹ (Molt4) and Rb⫺ (MB436) nonerythroid cell lines were
included as controls. Interestingly, the transformed counterpart of
FVA erythroblasts, MEL cells, had significantly reduced levels of
Figure 2. Expression of cell cycle–associated mRNAs during terminal erythroid
differentiation. Total RNA was isolated from FVA erythroblasts at the indicated times
during differentiation. Gel electrophoresis was performed, and ethidium bromidestained rRNA was photographed as a control for loading (A). Northern blot analysis
was performed using radiolabeled cDNA probes to Friend retroviral components (B),
␤-globin (C), cdk2 (D), cdk4 (E), cyclin D3 (F), and p21 (G).
erythroblasts, whereas abundant p21 was detected in p21 wild-type
erythroblasts treated with ActD. Levels of p27 protein also
increased during differentiation, albeit with different kinetics; p27
protein levels continued to increase, peaking at 48 rather than 24
hours. Contrary to the previous observation that cdk2, cdk4, and
cyclin D3 mRNA levels declined during differentiation, their
protein levels remained constant and were unaffected by the
absence of p21 or treatment with ActD. Levels of cdk6 and cyclin E
proteins, however, decreased during differentiation. Interestingly,
levels of cyclin E protein were greatly reduced in p21 null
erythroblasts and in p21 wild-type cells treated with ActD. Because
cyclin E protein levels are known to be positively regulated by
E2F1,26 it was not surprising that cyclin E protein levels were
reduced under experimental conditions in which ActD resulted in
the activation of p53 and the induction of p21. We have previously
shown that ActD-treated cells accumulate in G1,11 most likely
leading to the sequestration of functional E2F and the decreased
transcription of cyclin E. However, decreased cyclin E protein in
untreated p21 null erythroblasts was unexpected and remains to be
investigated further. Induction of p21 and p27 proteins during
Figure 3. Expression of cell cycle–associated proteins during terminal erythroid differentiation. Total cellular protein was isolated from FVA erythroblasts
cultured for 0, 24, or 48 hours with EPO (lanes 1-3), p21 null erythroblasts (lane 4), or
p21⫹/⫹ erythroblasts exposed to the DNA-damaging agent ActD for 6 hours (lane 5).
Proteins were electrophoretically separated in 12% polyacrylamide gels and probed
with antibodies to p21, p27, cdk2, cdk4, cdk6, cyclin D3, or cyclin E. gp55 protein
expression is shown as a control for protein loading.
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2750
HSIEH et al
Figure 4. Endogenous Rb protein expression during terminal erythroid differentiation. Steady state levels and the phosphorylation status of Rb protein were
determined by immunoblot analysis of whole cell lysates from FVA erythroblasts
cultured with or without EPO for the indicated times. Known Rb⫺ (MB436) and Rb⫹
(Molt 4) cell lines were included as controls. Equal amounts of protein were
electrophoretically separated in a 7% polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with anti-Rb. Positions of hyperphosphorylated (ppRb) and
hypophosphorylated (pRb) Rb are indicated. gp55 protein expression is shown as a
control for protein loading.
Rb compared to FVA erythroblasts, suggesting that the loss of Rb
protein may be associated with Friend virus-induced erythroleukemic transformation. Expression of the viral protein, gp55, was
evaluated as a control for erythroid cell protein loading. The data
are consistent with a G1 cell cycle arrest accompanied by an
accumulation of hypo-phosphorylated Rb during terminal erythroid differentiation.
Temporal regulation of G1 CDK kinase activity during terminal
erythroid differentiation
To determine which of the G1 CDKs may be associated with loss of
Rb phosphorylation in vivo, we performed in vitro Rb kinase
assays using immunoprecipitated CDK proteins (Figure 5). At the
initiation of culture, cdk6 kinase activity was abundant but was
extinguished very early in the time course. Cdk2 kinase activity
decreased gradually and was characterized by a marked decrease
by 24 hours and again by 48 hours. Cdk4 kinase activity, on the
other hand, remained constant in the first 24 hours of culture and
was abruptly extinguished late in the differentiation course. The
same cell lines as shown previously (Figure 4) were included as
controls. Although the amount of endogenous Rb protein was
significantly reduced in MEL cells than in their nontransformed
counterparts, FVA cells (Figure 4), the Rb kinase activity of all 3 G1
kinases tested in MEL cells was equivalent to or higher than that
observed in FVA cells. These data suggest that any deregulation of
Rb in MEL cells occurs downstream of G1 CDK kinase activity.
The kinetics of loss of CDK kinase activity demonstrated that G1
CDK kinase function is temporally regulated during terminal
erythroid differentiation.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
not detected in cdk2 immune complexes, but then appeared by 24
hours and persisted at 48 hours. No p21 protein was detected in the
cdk2 immune complex at any time. When cdk4 immunoprecipitations were examined for the presence of cdk4 protein (Figure 6B),
an unexpected result was observed. Levels of immunoprecipitatable cdk4 protein decreased significantly during the differentiation
time course, despite the fact that steady state levels of cdk4, as
determined by Western blot analysis (Figure 3D), remained unchanged. Unlike the observation in cdk2 immune complexes, p27
protein was detected in the cdk4 immune complex at the initiation
of culture, persisted at 24 hours, and was undetectable at 48 hours.
The absence of p27 in the complex at 48 hours was consistent with
the inability to immunoprecipitate cdk4 at that time. As observed in
cdk2 immunoprecipitations, no p21 protein was detected in cdk4
complexes at any time. In a separate experiment p21 was readily
detected in cdk2 and, to a lesser extent, in cdk4 immune complexes
after the treatment of cells with ActD (Figure 6D). The levels of
cdk6 protein in cdk6 immune complexes (Figure 6C) decreased
during differentiation, consistent with the observed decrease in
steady state levels of cdk6 (Figure 3E). At 0 and 24 hours, p27
protein was associated with cdk6 immune complexes. No p21 was
detected in cdk6 immune complexes during differentiation or in
cells treated with ActD. The data demonstrate that p27, but not p21,
binds to cdk4 and cdk6 early in differentiation and to cdk2 later in
differentiation and that p27 may be responsible for the observed
loss of Rb kinase activity in vivo and in vitro. The reason for the
inability to immunoprecipitate cdk4 at 48 hours was unclear until
experimental conditions were reversed and the p27 pool of protein was
immunoprecipitated first, followed by Western blot analysis
for CDKs.
p27 association with cdk4 and cdk2 is temporally distinct
during terminal erythroid differentiation
To further investigate the kinetics of p27/CDK associations during
differentiation, p27 immunoprecipitations were performed and
evaluated for the presence of cdk2, cdk4, cdk6, or cyclin D3
(Figure 7). At the initiation of culture, the small amount of p27
protein available was associated exclusively with cdk4. By 24
hours, when levels of p27 protein were significantly increased, the
amount of cdk4 associated with p27 increased proportionally.
p27, but not p21, associates with cdk4, cdk6, and cdk2 immune
complexes during terminal erythroid differentiation
To determine whether CDK kinase activity was differentially
regulated by p21, p27, or both during erythroid differentiation, we
performed CDK immunoprecipitations and examined the complexes for the presence of the Cip/Kip inhibitors. Western blot
analysis with anti-cdk2 (Figure 6A) demonstrated that levels of
cdk2 protein remained constant during differentiation. This finding
was consistent with the previous observation that steady state
levels of cdk2, as determined by Western blot analysis (Figure 3C),
remained unchanged. At the initiation of culture, p27 protein was
Figure 5. Rb kinase activity of CDK immunoprecipitates during terminal
erythroid differentiation. Whole cell lysates from FVA erythroblasts (0, 24, 48
hours) (lanes 1-3), murine erythroleukemia (MEL) (lane 4), MB436 (lane 5), and Molt
4 (lane 6) cells were immunoprecipitated with antibodies to cdk6, cdk2, or cdk4.
Immunoprecipitates were subjected to in vitro kinase assays using Rb as a substrate.
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
p27Kip1 INHIBITION OF cdk2 KINASE IN ERYTHROID
2751
Figure 6. CDK-immune complex formation during
terminal erythroid differentiation. Whole cell lysates
from FVA erythroblasts cultured with EPO for 0, 24, or
48 hours were immunoprecipitated with anti-cdk2 (A),
-cdk4 (B), or -cdk6 (C) in the absence (⫺) or presence
(⫹) of relevant blocking peptide. FVA erythroblasts
were cultured with EPO for 6 hours in the presence of
ActD and immunoprecipitated with anti-cdk2 or -cdk4
(D) or -cdk6 (C) in the absence (⫺) or presence (⫹) of
relevant blocking peptide. Immune complexes were
electrophoretically separated in 12% polyacrylamide
gels and transferred to PVDF membranes. Western
blot analysis was performed with antibodies to cdk2,
cdk4, cdk6, p27, or p21.
Despite the fact that a larger percentage of the total cdk4 protein
pool was now associated with p27, the in vitro cdk4 kinase activity
remained unchanged (Figure 5, lanes 1 and 2). No cdk2–p27
association was detected at 0 hours, but cdk2 protein was detected
in the p27 complex by 24 hours, when a significant decrease in in
vitro cdk2 kinase activity was observed (Figure 5, lanes 1 and 2).
After 48 hours of culture, abundant cdk4 protein was detected in
p27 immune complexes despite the fact that a cdk4–p27 association was not observed when the reciprocal experiment was
performed (Figure 6B). Cdk2 and cyclin D3 proteins were also
observed in p27 immune complexes at 48 h, when no cdk2 or cdk4
in vitro kinase activity was apparent (Figure 5, lane 3). Cdk6
protein was not detectable in p27 immune complexes at any time
(data not shown), suggesting that most of the p27 pool associates
with cdk4 rather than with cdk6.
INK4 expression during terminal erythroid differentiation
Figure 7. p27 immune complex formation during terminal erythroid differentiation. Whole cell lysates from FVA erythroblasts (0, 24, or 48 hours) were immunoprecipitated with anti-p27 in the absence (⫺) or presence (⫹) of blocking peptide.
Immune complexes were electrophoretically separated in 12% polyacrylamide gels
and transferred to PVDF membranes. Western blot analysis was performed with
antibodies to p27, cdk2, cdk4, or cyclin D3.
To provide a complete understanding of the regulatory proteins
associated with cell cycle exit of differentiating erythroblasts, we
examined expression of members of the INK4 family of CKIs.
Figure 8A represents total RNA isolated from 2 established
erythroid cell lines, HCD57 (EPO-dependent) and MEL cells
(EPO-independent), as well as undifferentiated FVA erythroblasts.
p16 mRNA was present in both cell lines but absent in FVA
erythroblasts. p19 and p18 mRNAs were present in all cells
examined, whereas a 1.3-kb mRNA species representing p15
(previously reported in certain mouse tissues, excluding spleen28)
was not detected in any of the cells tested. Figure 8B represents
expression of p19 protein during FVA erythroblast differentiation.
p19 protein levels increased during differentiation until 26 hours of
culture and then decreased markedly. We were unable to detect p18
protein at any time during differentiation (data not shown).
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2752
HSIEH et al
Figure 8. INK4 expression during terminal erythroid differentiation. (A) Total
RNA was isolated from HCD57, FVA, or MEL erythroblasts. Gel electrophoresis was
performed, and ethidium bromide–stained rRNA was photographed as a control for
loading. Northern blot analysis was performed using radiolabeled cDNA probes to
p16, p19, p18, and p15. (B) Total cellular protein was isolated from FVA erythroblasts
cultured for 0, 6, 14, 20, 26, 37, 42, or 48 hours with EPO (lanes 1-8). Proteins were
electrophoretically separated in 12% polyacrylamide gels and probed with antibodies
to p19. gp55 protein expression is shown as a control for protein loading.
BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
activity, the kinetics of p27 accumulation in cdk2 immune complexes
was consistent with the loss of cdk2 kinase activity and the growth arrest
of cells into G1. A model that describes the role of cdk2, cdk4, and p27 in
the growth arrest of differentiating cells is shown in Figure 9. At
initiation of the differentiation time course, low levels of p27 protein
associate primarily with cdk4. As differentiation proceeds, the amount
of p27 protein increases and p27/cdk4 complexes accumulate, with no
apparent adverse effect on cdk4 kinase activity. When cdk4 complexes
become saturated with p27, excess p27 protein is available to associate
with cdk2, in which it has a profound inhibitory effect on cdk2 kinase
activity. This model implies a switching mechanism whereby cdk4 (and
possibly cdk6) functions to sequester p27 as it accumulates in the cell
until the precise time that initiation of G1 arrest is indicated. Then excess p27 becomes available to inhibit cdk2/cyclin
E complexes.
Complete loss of cdk2 kinase activity does not occur until late
in differentiation and most likely results from additional events
distinct from p27 binding; the levels of associated p27/cdk2
proteins remained comparable at 24 and 48 hours (Figure 7) despite
marked differences in cdk2 kinase activity (Figure 5). Loss of
cyclin E from cdk2 complexes likely represents a late event
responsible for extinguishing cdk2 kinase activity in conjunction
with p27 binding. At 48 hours of culture, when cdk2 kinase activity
was largely extinguished (Figure 5), steady state cyclin E protein
levels were significantly reduced (Figure 3). Evidence supporting
the hypothesis that p27 binds cdk2 in the absence of cyclin E is
provided by studies in which in vitro translated CDK proteins bind
p27 in the absence of cyclins.29 These observations do not exclude
the possibility that other events, in addition to accumulation of p27
and loss of cyclin E proteins, are involved in regulating cdk2 kinase
function during differentiation.
The difference in kinetics of inhibition of cdk6 and cdk4 kinase
activity during differentiation suggests that these proteins are not
functionally redundant. Unlike Cdk6, Cdk4 activity was not
extinguished until very late in differentiation, presumably because
of an event other than p27 binding. One possibility is that a new
protein entered the complex and was responsible for the inhibition
of cdk4 kinase activity. INK4 family members are known to
specifically inhibit cyclin D–associated kinases. INK4 candidates
responsible for the inhibition of cdk4 or cdk6, or both, in FVA
erythroblasts include p19 and p18 proteins because p15 and p16
mRNA species were absent; p18 protein was undetectable in
Discussion
We used the FVA system as an experimental paradigm to study the
stopping mechanism associated with cell cycle exit of terminally
differentiating cells. In this model, primary erythroblasts differentiate in
response to EPO. Regulation of G1 cell cycle–associated proteins during
erythroid differentiation was primarily restricted to the induction of CKI
family members p21, p27, and p19 and the reduction of cdk6 and cyclin
E protein levels. Levels of the principal G1 CDK proteins, cdk4 and
cdk2, and the only cyclin D expressed in FVA erythroblasts, D3,
remained constant during differentiation. p27 protein accumulated
initially in cdk4 (and, to a lesser extent, in cdk6) immune complexes and
subsequently in cdk2 immune complexes. Although the association of
p27 with cdk4 had no apparent inhibitory effect on cdk4 in vitro kinase
Figure 9. Model of p27 function in terminal erythroid differentiation. p27 protein
accumulates in cyclin D/cdk4 complexes during differentiation without inhibiting
kinase activity. When cyclin D/cdk4 complexes become saturated with p27, p27
accumulates in cyclin E/cdk2 complexes and inhibits cdk2 kinase. Complete loss of
cdk2 kinase activity requires loss of cyclin E from the complex. Loss of cyclin D/cdk4
kinase activity occurs by an unknown mechanism.
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BLOOD, 15 OCTOBER 2000 䡠 VOLUME 96, NUMBER 8
differentiating FVA erythroblasts. Two different mRNA species
have been reported for p18 in mouse tissues, 2.2 and 1.1 kb.28 The
2.2-kb species, as detected in FVA erythroblasts, demonstrated
limited expression in other mouse tissues and may generate a
protein product for which the p18 antibody used does not react.
Alternatively, p18 protein may not be translated in FVA erythroblasts, or it may be expressed at levels below our limit of detection.
These possibilities require further investigation. p19 protein expression increased early in differentiation but was undetectable at later
time points, making it an unlikely candidate for inhibiting cdk4
kinase, though p19 inhibition of cdk6 kinase remains a possibility.
Alternatively, posttranslational modification of cdk4 or cdk6 or
some other existing member of the complexes may be associated
with the loss of kinase activity. Either of these mechanisms may be
responsible for the inability of cdk4 antibodies to recognize the
complex in immunoprecipitation experiments performed late in
differentiation. Further experiments will be required to identify the
precise mechanisms associated with inhibition of cdk4 and cdk6
kinases in terminal erythroid differentiation.
Our data demonstrating that p27 fails to inhibit the activity of
cyclin D–associated kinase, but not cyclin E-associated kinase, is
consistent with previously reported observations. First, all cyclin
D-CDK Rb kinase activity in proliferating mammalian cells is
found in complexes containing Cip/Kip proteins.30-36 Second, Soos
et al30 demonstrated that immunoprecipitation of p27 complexes
from a B-lymphocyte cell line contained kinase activity with
substrate specificity for Rb but not histone H1, a hallmark of cyclin
D–dependent kinase activity. In those studies, immunodepletion of
cdk6 removed most p27-associated kinase activity, implying that
p27 resided in a kinase-active cyclin D-cdk6–p27 complex. These
data suggest that under certain circumstances Cip/Kip proteins do
not inhibit cyclin D-dependent kinase activity in vivo and that
inhibition in vitro may depend on stoichiometry. Evidence supporting the latter comes from the observation that cyclin D-cdk6–p27/
p21 complexes that exist in a 1:1:1 ratio remain kinase active,31,32,37
whereas complexes containing a higher ratio of p21 or p27 proteins
are kinase inactive.38,39 On the contrary, equimolar concentrations
of p21 in cyclin E–cdk2 complexes were found to be inhibitory.40
Taken together, these studies demonstrate that Cip/Kip proteins are
functionally heterogeneous with regard to their ability to regulate
the activity of cyclin/CDK complexes.
Several reports have suggested a role for p27 in terminal
differentiation of other cell types, particularly oligodendrocytes.41-45 Durand et al44 demonstrated that p27-deficient oligodendrocytes proceeded through additional cell divisions before differentiation when compared to wild-type cells, suggesting that p27 is
part of a timing mechanism that determines when precursors stop
dividing and differentiate. These investigators45 also demonstrated
enhanced sensitivity of p27-null oligodendrocytes to various
mitogens.44 A role for p27 in erythroid differentiation is supported
by the observation that p27-deficient mice exhibit hyperplasia of
multiple organs, including the spleen,46-48 and have increased
numbers of early and late erythroid progenitor cells (BFU-E and
CFU-E) in the spleen and bone marrow.48 This observation
p27Kip1 INHIBITION OF cdk2 KINASE IN ERYTHROID
2753
suggests that the absence of p27 may allow for continued cell
proliferation, at the expense of differentiation, of early erythroid
progenitor cells.
p21 has been implicated in terminal differentiation of various
tissues. MyoD-induced expression of p21 in terminally differentiating muscle cells49 and forced overexpression of p21 in myoblasts
resulted in terminal differentiation,50 suggesting that p21 plays a
crucial role in muscle development. Further, examination of
p21-deficient keratinocytes showed a more rapid S phase and
reduced expression of a subset of differentiation markers.51 However, mice lacking p21 did not exhibit any developmental abnormalities, including muscle or skin,52 suggesting that other proteins
(perhaps p27) may substitute for p21 function. We did not detect a
role for p21 in the growth arrest associated with terminal erythroid
differentiation. Unlike p27, no p21 protein was found associated
with cdk2, cdk4, or cdk6 immune complexes. One possibility is
that p21 is not present in the same cellular compartment as G1 CDK
during erythroid differentiation. Alternatively, p21 may associate
with proteins other than G1 CDK during differentiation. Finally, it
remains possible that low levels of p21 did associate with one or all
G1 CDK complexes but were below our limits of detection. Our
data suggest that p21 may have a function other than growth arrest
during terminal erythroid differentiation. In a previous study we
reported that p21 protein levels were induced during differentiation
primarily through a p53-dependent mechanism.11 In that study we
were unable to detect a differentiation advantage in p53 wild-type
cells expressing abundant levels of p21 versus p53 null cells
expressing low levels of p21. Similarly, no differences were
observed in the ability of cells to accumulate into G1, regardless of
the p53 genotype and p21 protein levels. We speculated that
p53-dependent induction of p21 during erythroid differentiation
may represent a novel tumor suppressor function intended to
monitor normal differentiation in the event that differentiation is
blocked by activation of an oncogene or some other unforeseen
event. Ultimately, the use of erythroblasts derived from transgenic
animals lacking p21, p27, or both will be necessary to differentiate
the roles these gene products play in erythroid cell growth arrest
and terminal differentiation.
It remains to be determined how the growth arrest pathway is
linked to EPO signaling. Carroll et al53 have reported that the length
of G1 phase is altered in a murine EPO-dependent cell line, Ba/F3,
by culturing the cells in different concentrations of EPO, leading to
conditions that favored proliferation versus differentiation.53 Investigation of cell cycle regulators under these conditions should
provide insight into the molecular mechanisms of growth versus
differentiation.
Acknowledgment
We thank the Utah Regional Cancer Center for their core grant,
NCI 5P30CA42014, allowing us the use of the flow cytometry
facility.
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2000 96: 2746-2754
Cell cycle exit during terminal erythroid differentiation is associated with
accumulation of p27 Kip1 and inactivation of cdk2 kinase
Fen F. Hsieh, Lou Ann Barnett, Wayne F. Green, Karen Freedman, Igor Matushansky, Arthur I.
Skoultchi and Linda L. Kelley
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